Abstract: In order to prepare a composite hydrogel with nano-zinc oxide（ZnO NPs） and polyhexamethylene bisguanidine（PHMB） as the main drugs, in the experiment, the in vitro bacterial inhibition test was used to determine the minimum inhibitory concentration（MIC） and graded inhibitory concentration index（FIC） of the two drugs, and to investigate the optimal ratio of ZnO NPs and PHMB against Staphylococcus aureus. A one-way test was conducted to screen the matrix, matrix dosage, glycerol addition and PHMB mass concentration of ZnO NPs-PHMB composite hydrogel. Orthogonal experimental design was carried out based on a one-way test, and the ZnO NPs-PHMB composite hydrogel was prepared according to the optimized formulation; the quality of the ZnO NPs-PHMB composite hydrogel was evaluated in terms of its appearance, stability, viscosity, and antimicrobial properties. The results showed that the MIC of ZnO NPs and PHMB against Staphylococcus aureus were 64,0.5 μg/mL, respectively. The optimal prescription of ZnO NPs-PHMB composite hydrogel was chitosan quaternary ammonium salt 6.50%, glycerol 8.00%, PHMB 2.00 μg/mL, and ZnO NPs 8.00 μg/mL. The appearance of ZnO NPs-PHMB composite hydrogel was light yellow and transparent, pH value 5.18±0.50, viscosity 65.0 Pa·s, centrifugal stability and high and low temperature stability were good. The ZnO NPs-PHMB composite hydrogel had obvious inhibition effect on Staphylococcus aureus, and the inhibition rate of Staphylococcus aureus was 98.32%. The results suggested that the ZnO NPs-PHMB composite hydrogel was stable in quality and had good in vitro bacterial inhibition effect.